The authors would like to thank Ane M Rulykke for excellent technical assistance. We would like to thank Jesper Jurlander for sharing reagents and ideas. Anti-CD20 antibodies were a kind gift from Mark S. Cragg and Claude H.T. Chan, whom we would also like to thank for scientific discussions. We would like to thank Esben G. Schmidt for technical support and Morten Rasch for advice on protease inhibition. This work was made possible by the University of Copenhagen, Faculty of Health Sciences and The Neye Foundation. The authors declare to have no financial conflicts or interest. “
“Formation LGK-974 order of immune synapses (IS) between T cells and

APC requires multiple rearrangements in the actin cytoskeleton and selective receptor accumulation in supramolecular activation

clusters (SMAC). The inner cluster (central SMAC) contains the TCR/CD3 complex. The outer cluster (peripheral SMAC) contains the integrin LFA-1 and Talin. Molecular mechanisms selectively stabilizing receptors in the IS remained largely unknown. Here, we demonstrate that sustained LFA-1 clustering in the IS is a consequence of the combined activities of the actin-bundling protein L-plastin (LPL) and calmodulin. Thus, upon antigen-recognition of T cells, LPL accumulated predominantly in the peripheral SMAC. siRNA-mediated knock-down of LPL led to a failure of LFA-1 and Talin redistribution – however, not TCR/CD3 relocalization – into the IS. As a result of this LPL knock-down, the T-cell/APC interface became smaller over time and T-cell proliferation was inhibited. Importantly, mTOR inhibitor binding of calmodulin to LPL was required

for the maintenance of LPL in the IS and consequently inhibition of calmodulin also prevented stable accumulation of LFA-1 and Talin, but not CD3, in the IS. During the activation of T cells Obatoclax Mesylate (GX15-070) the immune synapse (IS) is formed at the area of interaction between T cells and APC 1, 2. The IS is involved in enhancing, directing and terminating the T-cell immune response (for review, see 3–7). Within the IS, surface receptors as well as intracellular signaling and scaffolding proteins are organized in distinct structures, which are called supramolecular activation clusters (SMAC). The inner cluster (central SMAC or cSMAC) contains PKCΘ and the TCR/CD3 complex. The outer cluster (peripheral SMAC or pSMAC) is composed of the integrin LFA-1 (CD11a/CD18) and Talin 8. It is clear that for the development of an IS the actin cytoskeleton is of special importance 2, 9–11. For construction of an actin meshwork, as it is found in the IS, crosslinking and bundling of F-actin is indispensable to support F-actin rigidity. Here, we demonstrate that the actin-bundling protein L-plastin (LPL) is an important component to orchestrate the ordered formation of a mature IS. LPL is a leukocyte-specific protein.